Microinjection of Follicle-Enclosed Mouse Oocytes..Pdf

Chapter 12 Microinjection of Follicle-Enclosed Mouse Oocytes Laurinda A. Jaffe, Rachael P. Norris, Marina Freudzon, William J. Ratzan, and Lisa M. Mehlmann Abstract The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle- enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment. Key words: Microinjection, follicle-enclosed oocyte, mouse. 1. Introduction Intracellular microinjection allows the study of biochemical events within intact cells, but since most cells are interconnected to form complex tissues, many physiological processes could most ideally be investigated by microinjection of cells within intact tissues. This chapter describes methods for injecting mouse oocytes within the complex of surrounding somatic cells known as the follicle (Fig. 12.1). These methods allow the introduction of molecules into the oocyte specifically, without also applying them to the somatic cells, and without disassembling the structure that is needed for investigating physiological responses. These approaches can be used to introduce antibodies, signaling proteins, dominant- negative proteins, buffers, or toxins that interfere with or mimic regulatory pathways (1–4), as well as optical probes to monitor signaling (3,5–7). Because follicles can be cultured for multiple days, these methods also allow the time needed for protein turnover following injection of short interfering RNAs (8). David J. Carroll (ed.), Microinjection: Methods and Applications, Vol. 518 Ó 2009 Humana Press, a part of Springer ScienceþBusiness Media, LLC DOI 10.1007/978-1-59745-202-1_12 157 158 Jaffe et al. Fig. 12.1. Developmental stages of mouse ovarian follicles. From ref. (10), # Society for Reproduction and Fertility (2005). Reproduced by permission. The mouse ovarian follicle forms as a single layer of somatic cells around the oocyte; the somatic cells then proliferate to form multiple layers (Fig. 12.1). Follicles containing an oocyte that is surrounded by 2–3 layers of somatic cells are referred to as ‘‘preantral’’. As the follicle continues to grow, spaces form between the somatic cells, and the spaces fuse to form a single antrum. The antrum separates the 1–3 layers of cumulus cells that directly surround the oocyte from the peripheral layers of mural granulosa cells; the cumulus mass is connected on one side to the mural cells. In response to follicle stimulating hormone (FSH), which stimulates follicle growth, receptors for luteinizing hormone (LH) are synthesized in the mural granulosa cells. Up to this point, the oocyte is arrested in meiotic prophase. Then, in response to a surge of LH from the pituitary, meiosis resumes and progresses to metaphase II, and ovulation occurs. This chapter describes methods for microinjecting follicle-enclosed oocytes at both preantral and antral stages. In our lab, we have used these methods to identify mechanisms by which meiotic arrest is maintained prior to the LH surge, and to investigate mechanisms by which LH stimulates meiotic resumption. Although cAMP in the oocyte had long been recog- nized as an inhibitor of meiotic progression (see refs (9,10)), it had been uncertain where and how this cAMP is generated. Micro- injection of follicle-enclosed oocytes using the methods described here has contributed essential evidence that a constitutively active G-protein coupled receptor (GPR3) activates Gs in the oocyte, leading to cAMP production (1, 2, 4, 6, 7). Studies using these methods have also provided evidence that LH does not cause meiotic resumption by terminating GPR3/Gs signaling (7),or by stimulating Gi signaling (3). The recent development of optical probes for monitoring cAMP dynamics in living cells (11) should, Microinjection of Follicle-Enclosed Mouse Oocytes 159 in combination with these microinjection techniques, allow further studies of the role of cAMP in regulating meiotic progression within an intact follicle. More generally, these techniques provide a new approach that could be used to investigate many other aspects of the complex bidirectional communication between the oocyte and somatic cells (12,13). The following methods for injection of follicle-enclosed mouse oocytes are based on methods that were previously devel- oped for microinjection of echinoderm oocytes (14–16).The follicle is placed between coverslips that compress it slightly, allowing the oocyte to be clearly visualized with a compound microscope (Fig. 12.2). The microinjection pipette is brought in horizontally, and contains mercury to allow precise control when pressure is applied through a screw-driven syringe. The pipette is front-loaded, and the volume injected is quantified by drawing up an equivalent volume of oil and then measuring the diameter of the expelled oil drop. These general methods have been described in detail in a recent chapter (16),which should be referred to for settinguptheequipment,andforthe basic procedures for injection. The present chapter explains how these techniques have been adapted for follicle-enclosed mouse oocytes. Videos demonstrating these procedures can be viewed at http://www.sciencedirect.com/science/MiamiMultiMediaURL/ B6WDG-4P8B0X8-6/B6WDG-4P8B0X8-6-C/6766/63fd3555 30400573e073a9091ba406e0/video1.mov (supplementary material for ref. 7), and at http://www.jcb.org/cgi/content/full/jcb. 200506194/DC1 (supplementary material for ref. 6). Fig. 12.2. Microinjection of follicle-enclosed mouse oocytes. (A) Diagram of the microinjection chamber and micropipette. (B) Photograph of a plastic slide for assembling the injection chamber. (C) Photograph of an antral follicle-enclosed oocyte as it appears in the injection chamber. 160 Jaffe et al. 2. Materials 2.1. Supplies for 1. Mice. Follicles are most easily dissected from ovaries of pre- Dissection and Culture pubertal mice, 22–25 days old. We use BL/6 Â SJL F1 mice of Follicles (The Jackson Laboratory, #100012). 2. Equine chorionic gonadotropin (pregnant mare serum gonadotropin). From the National Hormone and Peptide Program (http://www.humc.edu/hormones/), or various commercial sources. Store single-use aliquots (50 IU/ml, 500 ml) at –80°C. 3. MEM (Invitrogen, #12000-022). We make this medium from powder, adding 25-mM NaHCO3,75mg/ml penicillin G, and 50 mg/ml streptomycin sulfate, and store at 4°C for up to 2 weeks. Add serum and other supplements on day of use. 4. Penicillin G (Sigma, #PEN-K). Store powder at room temperature. 5. Streptomycin sulfate (Sigma, #S-6501). Store powder at 4°C. 6. Fetal bovine serum (Invitrogen, #16000-044, or other suppli- ers). Store aliquots at –80°C, and then at 4°C for up to 2 weeks. 7. Insulin-transferrin-sodium selenite (Sigma, #I1884), dissolved according to the manufacturer’s instructions to make a 100X stock. Store aliquots at –80°C, and then at 4°C for up to 2 weeks. 8. FSH (ovine), from the National Hormone and Peptide Pro- gram. Store single-use aliquots (10 mg/ml, 25 ml) at –80°C. 9. Steriflip sterile disposable vacuum filter units, 0.2-mm pore size, 50-ml volume (Millipore, #SCGP 005 25). 10. Miniforceps (Fine Science Tools, #11200-14). 11. Millicell culture plate inserts (Millipore, #PICMORG50). 12. Petri dishes (Falcon, #1008, 35 mm). 2.2. Equipment for 1. Stereoscope with an eyepiece micrometer (for dissection of Microinjection follicles and assembly of injection chambers). 2. Vibration free table (a table with a steel plate supported by rubber spacers, or an air table). 3. Compound microscope with a 20x objective and focusable eyepieces with a micrometer reticle. 4. Micromanipulator with X, Y, and Z controls. 5. Horizontal micropipette puller. 6. Screw-driven syringe connected to a micropipette holder by a piece of narrow tubing containing fluorocarbon oil (Sigma #F9880, Fluorinert FC-70). Microinjection of Follicle-Enclosed Mouse Oocytes 161 7. Machined plastic slides for assembling the injection chamber (see Fig. 12.2B). 8. For further details, see ref. (16). 2.3. Equipment for 1. Warm air blower (Nevtek air stream incubator, #ASI400) Temperature, 2. Electric thermometer (Physitemp Instruments, #BAT-12R, Humidity, and CO2 with an IT-18 temperature probe) Control 3. Fritted glass cylinder (Corning, #31770-500C, distributed by VWR Scientific Products) 4. Flowmeter (Gilmont, #GF-8321-2410, distributed by Bar- nant Company) 5. Translucent silicon rubber sheet (Reiss Manufacturing, Inc., 1/800 thick) 6. Magnetic rubber sheet (Custom-Magnets.com) 2.4. Supplies for 1. Diamond knife (Fine Science Tools, #10100-00), or ceramic Construction of Mouth knife (Fine Science Tools, #10025-45) Pipettes for Follicle 2. Translucent silicon rubber sheet (Reiss Manufacturing, Inc., Transfer 1/1600 thick) 3. Polyethylene tubing (Clay Adams, #PE-60, internal diameter 0.76 mm) 4. Mouth pipette assembly (Sigma, #A5177-5EA), see Note 1. 5. Glass capillaries (Drummond Scientific, #9-000-1061, o.d. ¼ 0.8 mm, i.d. ¼ 0.6 mm) 6. Syringe filter (Fisher, #09-719C, 25 mm, 0.2 mm) 7. Glass capillaries (Drummond Scientific, #2-000-100, 100 ml calibrated pipettes) 8. Sigmacote (Sigma, # SL2) 2.5. Supplies for 1. Coverslips, 22-mm square, #1.5. These should be cleaned Construction and before use (see ref. (16)). Assembly of Injection 2. Diamond pencil. This is an ordinary diamond-tipped glass Chambers marker, not the ‘‘diamond knife’’ referred to in Section 2.4.1. 3. Clear flexible plastic ruler, to be used as a guide for cutting coverslips. 4. Black Plexiglas work surface (from a machinist). 5. Double-sided tape (Scotch #137, previously called ‘‘double- stick’’ tape, office supply store). 6. Double-coated tape (Scotch #667, office supply store). 7. Small sharp scissors (Fine Science Tools, #14370-22, Moria 10.5 cm straight).

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